Compositions and methods for sirna inhibition of angiogenesis

ABSTRACT

RNA interference using small interfering RNAs which are specific for the vascular endothelial growth factor (VEGF) gene and the VEGF receptor genes Flt-1 and Flk-1/KDR inhibit expression of these genes. Diseases which involve angiogenesis stimulated by overexpression of VEGF, such as diabetic retinopathy, age related macular degeneration and many types of cancer, can be treated by administering the small interfering RNAs.

REFERENCE TO GOVERNMENT GRANT

The invention described herein was supported in part by NIH/NEI grantno. R01-EY10820, EY-13410 and EY12156. The U.S. government has certainrights in this invention.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.11/518,524 filed on Sep. 8, 2006, which is a continuation-in-part ofU.S. application Ser. No. 10/294,228 filed on Nov. 14, 2002, now U.S.Pat. No. 7,148,342, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/398,417, filed on Jul. 24, 2002; allaforementioned applications are incorporated by reference herein.

JOINT RESEARCH AGREEMENT

Not Applicable

FIELD OF THE INVENTION

Not Applicable

BACKGROUND OF THE INVENTION

Not Applicable

Angiogenesis, defined as the growth of new capillary blood vessels or“neovascularization,” plays a fundamental role in growth anddevelopment. In mature humans, the ability to initiate angiogenesis ispresent in all tissues, but is held under strict control. A keyregulator of angiogenesis is vascular endothelial growth factor(“VEGF”), also called vascular permeability factor (“VPF”). VEGF existsin at least four different alternative splice forms in humans (VEGF₁₂₁,VEGF₁₆₅, VEGF₁₈₉ and VEGF₂₀₆), all of which exert similar biologicalactivities.

Angiogenesis is initiated when secreted VEGF binds to the Flt-1 andFlk-1/KDR receptors (also called VEGF receptor 1 and VEGF receptor 2),which are expressed on the surface of endothelial cells. Flt-1 andFlk-1/KDR are transmembrane protein tyrosine kinases, and binding ofVEGF initiates a cell signal cascade resulting in the ultimateneovascularization in the surrounding tissue.

Aberrant angiogenesis, or the pathogenic growth of new blood vessels, isimplicated in a number of conditions. Among these conditions arediabetic retinopathy, diabetic macular edema (DME), psoriasis, exudativeor “wet” age-related macular degeneration (“ARMD”), rheumatoid arthritisand other inflammatory diseases, and most cancers. The diseasesassociated with these conditions exhibit abnormally high levels of VEGF,and generally show a high degree of vascularization or vascularpermeability.

ARMD in particular is a clinically important angiogenic disease. Thiscondition is characterized by choroidal neovascularization in one orboth eyes in aging individuals, and is the major cause of blindness inindustrialized countries.

Diabetic macular edema (DME), also called diabetic retinopathy, is acomplication of the chronically high blood sugar afflicting diabetics.It is caused by leakiness of retinal blood vessels and the growth of newblood vessels on the retina, optic nerve and the iris. The leaky bloodvessels result in swelling of the retina and visual loss. The new bloodvessels that grow on the optic nerve and retina can also bleed,resulting in severe visual loss. In addition, new blood vessels in theiris clog the drain of the eye and can result in extremely high pressurein the eye with accompanying intense pain and the potential loss of theeye. DME can affect almost anyone with diabetes. In general, the longersomeone has diabetes, the greater the risk of developing DME.Eventually, almost everyone with juvenile-onset diabetes will developsome symptoms of DME. Those who acquire diabetes later in life are alsoat risk of DME, although they are somewhat less likely to developadvanced DME.

A number of therapeutic strategies exist for inhibiting aberrantangiogenesis, which attempt to reduce the production or effect of VEGF.For example, anti-VEGF or anti-VEGF receptor antibodies (Kim E S et al.(2002), PNAS USA 99: 11399-11404), and soluble VEGF “traps” whichcompete with endothelial cell receptors for VEGF binding (Holash J etal. (2002), PNAS USA 99: 11393-11398) have been developed. ClassicalVEGF “antisense” or aptamer therapies directed against VEGF geneexpression have also been proposed (U.S. published application2001/0021772 of Uhlmann et al.). However, the anti-angiogenic agentsused in these therapies can produce only a stoichiometric reduction inVEGF or VEGF receptor, and the agents are typically overwhelmed by theabnormally high production of VEGF by the diseased tissue. The resultsachieved with available anti-angiogenic therapies have therefore beenunsatisfactory.

RNA interference (hereinafter “RNAi”) is a method ofpost-transcriptional gene regulation that is conserved throughout manyeukaryotic organisms. RNAi is induced by short (i.e., <30 nucleotide)double stranded RNA (“dsRNA”) molecules which are present in the cell(Fire A et al. (1998), Nature 391: 806-811). These short dsRNAmolecules, called “short interfering RNA” or “siRNA,” cause thedestruction of messenger RNAs (“mRNAs”) which share sequence homologywith the siRNA to within one nucleotide resolution (Elbashir S M et al.(2001), Genes Dev, 15: 188-200). It is believed that the siRNA and thetargeted mRNA bind to an “RNA-induced silencing complex” or “RISC”,which cleaves the targeted mRNA. The siRNA is apparently recycled muchlike a multiple-turnover enzyme, with 1 siRNA molecule capable ofinducing cleavage of approximately 1000 mRNA molecules. siRNA-mediatedRNAi degradation of an mRNA is therefore more effective than currentlyavailable technologies for inhibiting expression of a target gene.

What is needed, therefore, are agents which selectively inhibitexpression of VEGF or VEGF receptors in catalytic or sub-stoichiometricamounts.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed to siRNAs thatspecifically target and cause RNAi-induced degradation of mRNA fromVEGF, Flt-1 and Flk-1/KDR genes. The siRNA compounds and compositionsare used to inhibit angiogenesis, in particular for the treatment ofcancerous tumors, age-related macular degeneration, and other angiogenicdiseases.

Another embodiment of the present invention provides an isolated siRNAwhich targets human VEGF mRNA, human Flt-1 mRNA, human Flk-1/KDR mRNA,or an alternative splice form, mutant or cognate thereof. The siRNAcomprises a sense RNA strand and an antisense RNA strand which form anRNA duplex. The sense RNA strand comprises a nucleotide sequenceidentical to a target sequence of about 19 to about 25 contiguousnucleotides in the target mRNA.

In another embodiment of the present invention, recombinant plasmids andviral vectors which express the siRNA, as well as pharmaceuticalcompositions comprising the siRNA and a pharmaceutically acceptablecarrier are provided.

Further embodiments of the present invention provide methods ofinhibiting expression of human VEGF mRNA, human Flt-1 mRNA, humanFlk-1/KDR mRNA, or an alternative splice form, mutant or cognatethereof, comprising administering to u subject an effective amount ofthe siRNA such that the target mRNA is degraded.

Other embodiments of the present invention provide methods of inhibitingangiogenesis and treating angiogenic diseases in a subject, comprisingadministering to a subject an effective amount of an siRNA targeted tohuman VEGF mRNA, human Flt-1 mRNA, human Flk-1/KDR mRNA, or analternative splice form, mutant or cognate thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a histograms of VEGF concentration (in pg/ml) inhypoxic 293 and HeLa cells treated with no siRNA (“-”); nonspecificsiRNA (“nonspecific”); or siRNA targeting human VEGF mRNA (“VEGF”). VEGFconcentration (in pg/ml) in non-hypoxic 293 and HeLa cells is alsoshown. Each bar represents the average of four experiments, and theerror is the standard deviation of the mean.

FIG. 2 is a histogram of murine VEGF concentration (in pg/ml) in hypoxicNIH 3T3 cells treated with no siRNA (“-”); nonspecific siRNA(“nonspecific”); or siRNA targeting human VEGF mRNA (“VEGF”). Each barrepresents the average of six experiments and the error is the standarddeviation of the mean.

FIG. 3 is a histogram of human VEGF concentration (pg/total protein) inretinas from mice injected with adenovirus expressing human VEGF(“AdVEGF”) in the presence of either GFP siRNA (dark gray bar) or humanVEGF siRNA (light grey bar). Each bar represents the average of 5 eyesand the error bars represent the standard error of the mean.

FIG. 4 is a histogram showing the mean area (in mm²) of laser-inducedCNV in control eyes given subretinal injections of GFP siRNA (N=9; “GFPsiRNA”), and in eyes given subretinal injections of mouse VEGF siRNA(N=7; “Mouse VEGF siRNA”). The error bars represent the standard errorof the mean.

FIG. 5 is a schematic representation of pAAVsiRNA, a cis-acting plasmidused to generate a recombinant AAV viral vector of the invention. “ITR”:AAV inverted terminal repeats; “U6”: U6 RNA promoters; “Sense”: siRNAsense coding sequence; “Anti”: siRNA antisense coding sequence; “PotyT”:polythymidine termination signals.

FIG. 6 shows histograms of the mean area (in mm²) of laser-induced CNVin treatment in mouse eyes injected (A) subretinally or (B)intravitreally with a mouse anti-VEGF siRNA (“mVEGF1.siRNA”) or controlsiRNA (“GFP1.siRNA”). The error bars represent the standard error of themean. (C) is a histogram of the mean area (in mm²) of laser-induced CNVin mouse eyes injected intravitreally with: phosphate-buffered salinewith no siRNA at 1 day post-laser induction (“PBS”; CNV area measured at14 days post-laser induction); control siRNA at 14 days post-laserinduction (“GFP1.siRNA”; CNV area measured at 21 days post-laserinduction); or a mouse anti-VEGF siRNA at 14 days post-laser induction(“mVEGF1.siRNA”; CNV area measured at 21 days post-laser induction). Theerror bars represent the standard error of the mean.

FIG. 7 is a graph of the percent of VEGF (“% VEGF”) protein in mouseeyes injected sub-retinally with human anti-VEGF siRNA (“Cand5”) andcontrol siRNA (“GFP1.siRNA”) at 0 (n=2; pre-siRNA injection), 6 (n=3),10 (n=3) and 14 (n=3) days post-injection. % VEGF=([VEGF] in the Cand5eye/[VEGF] in the GFP1.siRNA eye)*100.

FIG. 8 is a graph of hVEGF protein levels in 293 cells transfected withtransfected with human VEGF siRNAs, non-specific siRNA (EGFP siRNA) ormock transfections without siRNA.

FIG. 9 is a graph of the dose response studies with Cand5, hVEGF#1,hVEGF#2, hVEGF#3, hVEGF#4, hVEGF#6 and hVEGF#7.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “molecule” is a reference to one or more molecules and equivalentsthereof known to those skilled in the art, and so forth. As used herein,the term “about” means plus or minus 10% of the numerical value of thenumber with which it is being used. Therefore, about 50% means in therange of 45%-55%.

As used herein, a “subject” includes a human being or non-human animal.Preferably, the subject is a human being.

As used herein, an “effective amount” of the siRNA is an amountsufficient to cause RNAi-mediated degradation of the target mRNA, or anamount sufficient to inhibit the progression of angiogenesis in asubject.

As used herein, “isolated” means altered or removed from the naturalstate through human intervention. For example, an siRNA naturallypresent in a living animal is not “isolated,” but a synthetic siRNA, oran siRNA partially or completely separated from the coexisting materialsof its natural state is “isolated.” An isolated siRNA can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a cell into which the siRNA has been delivered.

As used herein, “target mRNA” means human VEGF, Flt-1 or Flk-1/KDR mRNA,mutant or alternative splice forms of human VEGF, Flt-1 or Flk-1/KDRmRNA, or mRNA from cognate VEGF, Flt-1 or Flk-1/KDR genes.

As used herein, a gene or mRNA which is “cognate” to human VEGF, Flt-1or Flk-1/KDR is a gene or mRNA from another mammalian species which ishomologous to human VEGF, Flt-1 or Flk-1/KDR. For example, the cognateVEGF mRNA from the mouse is given in SEQ ID NO: 1.

Unless otherwise indicated, all nucleic acid sequences herein are givenin the 5′ to 3′ direction. Also, all deoxyribonucleotides in a nucleicacid sequence are represented by capital letters (e.g., deoxythymidineis “T”), and ribonucleotides in a nucleic acid sequence are representedby lower case letters (e.g., uridine is “u”).

Compositions and methods comprising siRNA targeted to VEGF, Flt-1 orFlk-1/KDR mRNA are advantageously used to inhibit angiogenesis, inparticular for the treatment of angiogenic disease. The siRNA arebelieved to cause the RNAi-mediated degradation of these mRNAs, so thatthe protein product of the VEGF, Flt-1 or Flk-1/KDR genes is notproduced or is produced in reduced amounts. Because VEGF binding to theFlt-1 or Flk-1/KDR receptors is required for initiating and maintainingangiogenesis, the siRNA-mediated degradation of VEGF, Flt-1 or Flk-1/KDRmRNA inhibits the angiogenic process.

One aspect of the present invention therefore provides isolated siRNAcomprising short double-stranded RNA from about 17 nucleotides to about29 nucleotides in length, preferably from about 19 to about 25nucleotides in length, that are targeted to the target mRNA. The siRNAcomprise a sense RNA strand and a complementary antisense RNA strandannealed together by standard Watson-Crick base-pairing interactions(hereinafter “base-paired”). As is described in more detail below, thesense strand comprises a nucleic acid sequence which is identical to atarget sequence contained within the target mRNA.

The sense and antisense strands of the siRNA can comprise twocomplementary, single-stranded RNA molecules or can comprise a singlemolecule in which two complementary portions are base-paired and arecovalently linked by a single-stranded “hairpin” area. Without wishingto be bound by any theory, it is believed that the hairpin area of thelatter type of siRNA molecule is cleaved intracellularly by the “Dicer”protein (or its equivalent) to form an siRNA of two individualbase-paired RNA molecules (see Tuschl, T. (2002), supra).

Splice variants of human VEGF are known, including VEGF₁₂₁ (SEQ ID NO:2), VEGF₁₆₅ (SEQ ID NO: 3), VEGF₁₈₉ (SEQ ID NO: 4) and VEGF₂₀₆ (SEQ IDNO: 5). The mRNA transcribed from the human VEGF, Flt-1 (SEQ ID NO: 6)or Flk-1/KDR (SEQ ID NO: 7) genes can be analyzed for furtheralternative splice forms using techniques well-known in the art. Suchtechniques include reverse transcription-polymerase chain reaction(RT-PCR), northern blotting and in-situ hybridization. Techniques foranalyzing mRNA sequences are described, for example, in Busting SA(2000), J. Mol. Endocrinol. 25: 169-193, the entire disclosure of whichis herein incorporated by reference. Representative techniques foridentifying alternatively spliced mRNAs are also described below.

For example, databases that contain nucleotide sequences related to agiven disease gene can be used to identify alternatively spliced mRNA.Such databases include GenBank, Embase, and the Cancer Genome AnatomyProject (CGAP) database. The CGAP database, for example, containsexpressed sequence tags (ESTs) from various types of human cancers. AnmRNA or gene sequence from the VEGF, Flt-1 or Flk-1/KDR genes can beused to query such a database to determine whether ESTs representingalternatively spliced mRNAs have been found for a these genes.

A technique called “RNAse protection” can also be used to identifyalternatively spliced VEGF, Flt-1 or Flk-1/KDR mRNAs. RNAse protectioninvolves translation of a gene sequence into synthetic RNA, which ishybridized to RNA derived from other cells; for example, cells fromtissue at or near the site of neovascularization. The hybridized RNA isthen incubated with enzymes that recognize RNA:RNA hybrid mismatches.Smaller than expected fragments indicate the presence of alternativelyspliced mRNAs. The putative alternatively spliced mRNAs can be clonedand sequenced by methods well known to those skilled in the art.

RT-PCR can also be used to identify alternatively spliced VEGF, Flt-1 orFlk-1/KDR mRNAs. In RT-PCR, mRNA from the diseased tissue is convertedinto cDNA by the enzyme reverse transcriptase, using methods well-knownto those of ordinary skill in the art. The entire coding sequence of thecDNA is then amplified via PCR using a forward primer located in the 3′untranslated region, and a reverse primer located in the 5′ untranslatedregion. The amplified products can be analyzed for alternative spliceforms, for example by comparing the size of the amplified products withthe size of the expected product from normally spliced mRNA, e.g., byagarose gel electrophoresis. Any change in the size of the amplifiedproduct can indicate alternative splicing.

mRNA produced from mutant VEGF, Flt-1 or Flk-1/KDR genes can also bereadily identified through the techniques described above foridentifying alternative splice forms. As used herein, “mutant” VEGF,Flt-1 or Flk-1/KDR genes or mRNA include human VEGF, Flt-1 or Flk-1/KDRgenes or mRNA which differ in sequence from the VEGF, Flt-1 or Flk-1/KDRsequences set forth herein. Thus, allelic forms of these genes, and themRNA produced from them, are considered “mutants” for purposes of thisinvention.

It is understood that human VEGF, Flt-1 or Flk-1/KDR mRNA may containtarget sequences in common with their respective alternative spliceforms, cognates or mutants. A single siRNA comprising such a commontargeting sequence can therefore induce RNAi-mediated degradation ofdifferent RNA types which contain the common targeting sequence.

The siRNA can comprise partially purified RNA, substantially pure RNA,synthetic RNA, or recombinantly produced RNA, as well as altered RNAthat differs from naturally-occurring RNA by the addition, deletion,substitution and/or alteration of one or more nucleotides. Suchalterations can include addition of non-nucleotide material, such as tothe end(s) of the siRNA or to one or more internal nucleotides of thesiRNA, including modifications that make the siRNA resistant to nucleasedigestion.

One or both strands of the siRNA can also comprise a 3′ overhang. Asused herein, a “3′ overhang” refers to at least one unpaired nucleotideextending from the 3′-end of a duplexed RNA strand.

Thus in one embodiment, the siRNA comprises at least one 3′ overhang offrom 1 to about 6 nucleotides (which includes ribonucleotides ordeoxynucleotides) in length, preferably from 1 to about 5 nucleotides inlength, more preferably from 1 to about 4 nucleotides in length, andparticularly preferably from about 2 to about 4 nucleotides in length.

In the embodiment in which both strands of the siRNA molecule comprise a3′ overhang, the length of the overhangs can be the same or differentfor each strand. In a most preferred embodiment, the 3′ overhang ispresent on both strands of the siRNA, and is 2 nucleotides in length.For example, each strand of the siRNA can comprise 3′ overhangs ofdithymidylic acid (“TT”) or diuridylic acid (“uu”).

In order to enhance the stability of the present siRNA, the 3′ overhangscan be also stabilized against degradation. In one embodiment, theoverhangs are stabilized by including purine nucleotides, such asadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g., substitution ofuridine nucleotides in the 3′ overhangs with 2′-deoxythymidine, istolerated and does not affect the efficiency of RNAi degradation. Inparticular, the absence of a 2′ hydroxyl in the 2% deoxythymidinesignificantly enhances the nuclease resistance of the 3 ′ overhang intissue culture medium.

In certain embodiments, the siRNA comprises the sequence AA(N19)TT orNA(N21), where N is any nucleotide. These siRNA comprise approximately30-70% GC, and preferably comprise approximately 50% G/C. The sequenceof the sense siRNA strand corresponds to (N19)TT or N21 (i.e., positions3 to 23), respectively. In the latter case, the 3′ end of the sensesiRNA is converted to TT. The rationale for this sequence conversion isto generate a symmetric duplex with respect to the sequence compositionof the sense and antisense strand 3′ overhangs. The antisense RNA strandis then synthesized as the complement to positions 1 to 21 of the sensestrand.

Because position 1 of the 23-nt sense strand in these embodiments is notrecognized in a sequence-specific manner by the antisense strand, the3′-most nucleotide residue of the antisense strand can be chosendeliberately. However, the penultimate nucleotide of the antisensestrand (complementary to position 2 of the 23-nt sense strand in eitherembodiment) is generally complementary to the targeted sequence.

In another embodiment, the siRNA comprises the sequence NAR(N17)YNN,where R is a purine (e.g., A or G) and Y is a pyrimidine (e.g., C orU/T). The respective 21-nt sense and antisense RNA strands of thisembodiment therefore generally begin with a purine nucleotide. SuchsiRNA can be expressed from pol III expression vectors without a changein targeting site, as expression of RNAs from pol III promoters is onlybelieved to be efficient when the first transcribed nucleotide is apurine.

In a further embodiment, the siRNA comprises a sequence having no morethan five (5) consecutive purines or pyrimidines. In a furtherembodiment, the siRNA comprises a sequence having no more than five (5)consecutive nucleotides having the same nucleobase (i.e., A, C, G, orU/T).

The siRNA can be targeted to any stretch of approximately 19-25contiguous nucleotides in any of the target mRNA sequences (the “targetsequence”). Techniques for selecting target sequences for siRNA aregiven, for example, in Tuschl T et al., “The siRNA User Guide,” revisedOct. 11, 2002, the entire disclosure of which is herein incorporated byreference. “The siRNA User Guide” is available on the world wide web ata website maintained by Dr. Thomas Tuschl, Department of CellularBiochemistry, AG 105, Max-Planck-Institute for Biophysical Chemistry,37077 Göttingen, Germany, and can be found by accessing the website ofthe Max Planck Institute and searching with the keyword “siRNA.” Thus,the sense strand of the present siRNA comprises a nucleotide sequenceidentical to any contiguous stretch of about 19 to about 25 nucleotidesin the target mRNA.

Generally, a target sequence on the target mRNA can be selected from agiven cDNA sequence corresponding to the target mRNA, preferablybeginning 50 to 100 nt downstream (i.e., in the 3′ direction) from thestart codon. The target sequence can, however, be located in the 5′ or3′ untranslated regions, or in the region nearby the start codon (see,e.g., the target sequences of SEQ ID NOS: 73 and 74 in Table 1 below,which are within 0.100 nt of the 5′-end of the VEGF₁₂₁ cDNA.

In a further embodiment of the present invention, the target mRNAsequence comprises no more than five (5) consecutive purines orpyrimidines. For example, a suitable target sequence in the VEGF₁₂₁ cDNAsequence is:

TCATCACGAAGTGGTGAAG (SEQ ID NO: 8)

Thus, an siRNA targeting this sequence, and which has 3′ uu overhangs oneach strand (overhangs shown in bold), is:

5′-ucaucacgaaguggugaaguu-3′ (SEQ ID NO: 9) 3′-uuaguagugcuucaccacuuc-5′(SEQ ID NO: 10)

An siRNA targeting this same sequence, but having 3′ TT overhangs oneach strand (overhangs shown in bold) is:

5′-ucaucacgaaguggugaagTT-3′ (SEQ ID NO: 11) 3′-TTaguagugcuucaccacuuc-5′(SEQ ID NO: 12)

Other VEGF₁₂₁ target sequences from which siRNA can be derived are givenin Table 1. It is understood that all VEGF₁₂₁ target sequences listed inTable 1 are within that portion of the VEGF₁₂₁ alternative splice formwhich is common to all human VEGF alternative splice forms. Thus, theVEGF₁₂, target sequences in Table 1 can also target VEGF₁₆₅, VEGF₁₈₉ andVEGF₂₀₆ mRNA. Target sequences which target a specific VEGF isoform canalso be readily identified. For example, a target sequence which targetsVEGF₁₆₅ mRNA but not VEGF₁₂₁ mRNA is AACGTACTTGCAGATGTGACA (SEQ ID NO:13). Exemplary target sequences for human Flt-1 for human Flk-1/KDR aregiven in PCT/US2003/0022444 filed Jul. 18, 2003, herein incorporated byreference in its entirety.

TABLE 1 VEGF Target Sequences target sequence SEQ ID NO: cognate VEGFmRNA sequence  1 Splice variant VEGF₁₂₁ sequence  2 Splice variantVEGF₁₆₅ sequence  3 Splice variant VEGF₁₈₉ sequence  4 Splice variantVEGF₂₀₆ sequence  5 TCATCACGAAGTGGTGAAG  8 ucaucacgaaguggugaaguu  9uuaguagugcuucaccacuuc 10 ucaucacgaaguggugaagTT 11 TTaguagugcuucaccacuuc12 AACGTACTTGCAGATGTGACA 13 GTTCATGGATGTCTATCAG 14 TCGAGACCCTGGTGGACAT15 TGACGAGGGCCTGGAGTGT 16 TGACGAGGGCCTGGAGTGT 17 CATCACCATGCAGATTATG 18ACCTCACCAAGGCCAGCAC 19 GGCCAGCACATAGGAGAGA 20 CAAATGTGAATGCAGACCA 21ATGTGAATGCAGACCAAAG 22 TGCAGACCAAAGAAAGATA 23 AGAAAGATAGAGCAAGACA 24GAAAGATAGAGCAAGACAA 25 GATAGAGCAAGACAAGAAA 26 GACAAGAAAATCCCTGTGG 27GAAAATCCCTGTGGGCCTT 28 AATCCCTGTGGGCCTTGCT 29 TCCCTGTGGGCCTTGCTCA 30GCATTTGTTTGTACAAGAT 31 GATCCGCAGACGTGTAAAT 32 ATGTTCCTGCAAAAACACA 33TGTTCCTGCAAAAACACAG 34 AAACACAGACTCGCGTTGC 35 AACACAGACTCGCGTTGCA 36ACACAGACTCGCGTTGCAA 37 CACAGACTCGCGTTGCAAG 38 GGCGAGGCAGCTTGAGTTA 39ACGAACGTACTTGCAGATG 40 CGAACGTACTTGCAGATGT 41 CGTACTTGCAGATGTGACA 42GTGGTCCCAGGCTGCACCC 43 GGAGGAGGGCAGAATCATC 44 GTGGTGAAGTTCATGGATG 45AATCATCACGAAGTGGTGAAG 46 AAGTTCATGGATGTCTATCAG 47 AATCGAGACCCTGGTGGACAT48 AATGACGAGGGCCTGGAGTGT 49 AACATCACCATGCAGATTATG 50AAACCTCACCAAGGCCAGCAC 51 AAGGCCAGCACATAGGAGAGA 52 AACAAATGTGAATGCAGACCA53 AAATGTGAATGCAGACCAAAG 54 AATGCAGACCAAAGAAAGATA 55AAAGAAAGATAGAGCAAGACA 56 AAGAAAGATAGAGCAAGACAA 57AAGATAGAGCAAGACAAGAAAAT 58 AAGACAAGAAAATCCCTGTGGGC 59AAGAAAATCCCTGTGGGCCTTGC 60 AATCCCTGTGGGCCTTGCTCAGA 61AAGCATTTGTTTGTACAAGATCC 62 AAGATCCGCAGACGTGTAAATGT 63AAATGTTCCTGCAAAAACACAGA 64 AATGTTCCTGCAAAAACACAGAC 65AAAAACACAGACTCGCGTTGCAA 66 AAAACACAGACTCGCGTTGCAAG 67AAACACAGACTCGCGTTGCAAGG 68 AACACAGACTCGCGTTGCAAGGC 69AAGGCGAGGCAGCTTGAGTTAAA 70 AAACGAACGTACTTGCAGATGTG 71AACGAACGTACTTGCAGATGTGA 72 AAGTGGTCCCAGGCTGCACCCAT 73AAGGAGGAGGGCAGAATCATCAC 74 AAGTGGTGAAGTTCATGGATGTC 75AAAATCCCTGTGGGCCTTGCTCA 76 accucaccaaggccagcacTT 77gugcuggccuuggugagguTT 78 GGCTACGTCCAGCGCACC 79 AAACCUCACCAAAGCCAGCAC 80

The siRNA can be obtained using a number of techniques known to those ofskill in the art. For example, the siRNA can be chemically synthesizedor recombinantly produced using methods known in the art, such as theDrosophila in vitro system described in U.S. published application2002/0086356 of Tuschl et al., the entire disclosure of which is hereinincorporated by reference.

Preferably, the siRNA are chemically synthesized using appropriatelyprotected ribonucleoside phosphoramidites and a conventional DNA/RNAsynthesizer. The siRNA can be synthesized as two separate, complementaryRNA molecules, or as a single RNA molecule with two complementaryregions. Commercial suppliers of synthetic RNA molecules or synthesisreagents include Proligo (Hamburg, Germany), Dharmacon Research(Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science,Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes(Ashland, Mass., USA) and Cruachem (Glasgow, UK). The siRNA can also besynthesized as multiple complementary RNA molecules, as described inmore detail in co-pending U.S. Provisional Application No. ______,entitled “siRNA and Methods of Manufacture” filed simultaneouslyherewith, herein incorporated by reference in its entirety.

Alternatively, siRNA can also be expressed from recombinant circular orlinear DNA plasmids using any suitable promoter. Suitable promoters forexpressing siRNA from a plasmid include, for example, the U6 or H1 RNApol III promoter sequences and the cytomegalovirus promoter. Selectionof other suitable promoters is within the skill in the art. Therecombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the siRNA in a particular tissueor in a particular intracellular environment.

The siRNA expressed from recombinant plasmids can either be isolatedfrom cultured cell expression systems by standard techniques, or can beexpressed intracellularly at or near the area of neovascularization invivo. The use of recombinant plasmids to deliver siRNA to cells in vivois discussed in more detail below.

siRNA can be expressed from a recombinant plasmid either as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions.

Selection of plasmids suitable for expressing siRNA, methods forinserting nucleic acid sequences for expressing the siRNA into theplasmid, and methods of delivering the recombinant plasmid to the cellsof interest are within the skill in the art. See, for example Tuschl, T.(2002), Nat. Biotechnol, 20: 446-448; Brummelkamp TR et al. (2002),Science 296: 550-553; Miyagishi M et al. (2002), Nat. Biotechnol. 20:497-500; Paddison P J et al. (2002), Genes Dev. 16: 948-958; Lee N S etal. (2002), Nat. Biotechnol. 20: 500-505; and Paul C P et al. (2002),Nat. Biotechnol. 20: 505-508, the entire disclosures of which are hereinincorporated by reference.

A plasmid comprising nucleic acid sequences for expressing an siRNA isdescribed in Example 7 below. That plasmid, called pAAVsiRNA, comprisesa sense RNA strand coding sequence in operable connection with a polyTtermination sequence under the control of a human U6 RNA promoter, andan antisense RNA strand coding sequence in operable connection with apolyT termination sequence under the control of a human U6 RNA promoter.The plasmid pAAVsiRNA is ultimately intended for use in producing anrecombinant adeno-associated viral vector comprising the same nucleicacid sequences for expressing an siRNA.

As used herein, “in operable connection with a polyT terminationsequence” means that the nucleic acid sequences encoding the sense orantisense strands are immediately adjacent to the polyT terminationsignal in the 5′ direction. During transcription of the sense orantisense sequences from the plasmid, the polyT termination signals actto terminate transcription.

As used herein, “under the control” of a promoter means that the nucleicacid sequences encoding the sense or antisense strands are located 3′ ofthe promoter, so that the promoter can initiate transcription of thesense or antisense coding sequences.

The siRNA can also be expressed from recombinant viral vectorsintracellularly at or near the area of neovascularization in vivo. Therecombinant viral vectors of the invention comprise sequences encodingthe siRNA and any suitable promoter for expressing the siRNA sequences.Suitable promoters include, for example, the U6 or H1 RNA pol IIIpromoter sequences and the cytomegalovirus promoter. Selection of othersuitable promoters is within the skill in the art. The recombinant viralvectors of the invention can also comprise inducible or regulatablepromoters for expression of the siRNA in a particular tissue or in aparticular intracellular environment. The use of recombinant viralvectors to deliver siRNA to cells in vivo is discussed in more detailbelow.

siRNA can be expressed from a recombinant viral vector either as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions.

Any viral vector capable of accepting the coding sequences for the siRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of the viral vectors can also be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses. For example, an AAV vector of the inventioncan be pseudotyped with surface proteins from vesicular stomatitis virus(VSV), rabies, Ebola, Mokola, and the like.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe siRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller AD (1990), Hum Gene Therap. 1: 5-14;and Anderson WF (1998), Nature 392: 25-30, the entire disclosures ofwhich are herein incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the siRNA is expressed as twoseparate, complementary single-stranded RNA molecules from a recombinantAAV vector comprising, for example, either the U6 or H1 RNA promoters,or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the siRNA, a method for constructingthe recombinant. AV vector, and a method for delivering the vector intotarget cells, are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

Suitable AAV vectors for expressing the siRNA, methods for constructingthe recombinant AAV vector, and methods for delivering the vectors intotarget cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher K J et al. (1996), J. Virol., 70: 520-532; Samulski Ret al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S.Pat. No. 5,139,941; International Patent Application No. WO 94/13788;and International Patent Application No. WO 93/24641, the entiredisclosures of which are herein incorporated by reference. An exemplarymethod for generating a recombinant AAV vector of the invention isdescribed in Example 7 below.

The ability of an siRNA containing a given target sequence to causeRNAi-mediated degradation of the target mRNA can be evaluated usingstandard techniques for measuring the levels of RNA or protein in cells.For example, siRNA can be delivered to cultured cells, and the levels oftarget mRNA can be measured by Northern blot or dot blotting techniques,or by quantitative RT-PCR. Alternatively, the levels of VEGF, Flt-1 orFlk-1/KDR receptor protein in the cultured cells can be measured byELISA or Western blot. A suitable cell culture system for measuring theeffect of the present siRNA on target mRNA or protein levels isdescribed in Example 1 below.

RNAi-mediated degradation of target mRNA by an siRNA containing a giventarget sequence can also be evaluated with animal models ofneovascularization, such as the ROP or CNV mouse models. For example,areas of neovascularization in an ROP or CNV mouse can be measuredbefore and after administration of an siRNA. A reduction in the areas ofneovascularization in these models upon administration of the siRNAindicates the down-regulation of the target mRNA (see Example 6 below).

As discussed above, the siRNA is capable of targeting and causing theRNAi-mediated degradation of VEGF, Flt-1 or Flk-1/KDR mRNA, oralternative splice forms, mutants or cognates thereof, preferably VEGF,and more preferably human VEGF. Degradation of the target mRNA by thepresent siRNA reduces the production of a functional gene product fromthe VEGF, Flt-1 or Flk-1/KDR genes. Thus, another embodiment of thepresent invention provides a method of inhibiting expression of VEGF,Flt-1 or Flk-1/KDR in a subject, comprising administering an effectiveamount of an siRNA to the subject, such that the target mRNA isdegraded. As the products of the VEGF, Flt-1 and Flk-1/KDR genes arerequired for initiating and maintaining angiogenesis, another embodimentof the present invention provides a method of inhibiting angiogenesis ina subject by the RNAi-mediated degradation of the target mRNA by thepresent siRNA.

RNAi-mediated degradation of the target mRNA can be detected bymeasuring levels of the target mRNA or protein in the cells of asubject, using standard techniques for isolating and quantifying mRNA orprotein as described above.

Inhibition of angiogenesis can be evaluated by directly measuring theprogress of pathogenic or nonpathogenic angiogenesis in a subject; forexample, by observing the size of a neovascularized area before andafter treatment with the siRNA. An inhibition of angiogenesis isindicated if the size of the neovascularized area stays the same or isreduced. Techniques for observing and measuring the size ofneovascularized areas in a subject are within the skill in the art; forexample, areas of choroid neovascularization can be observed byopthalmoscopy.

Inhibition of angiogenesis can also be inferred through observing achange or reversal in a pathogenic condition associated with theangiogenesis. For example, in ARMD, a slowing, halting or reversal ofvision loss indicates an inhibition of angiogenesis in the choroid. Fortumors, a slowing, halting or reversal of tumor growth, or a slowing orhalting of tumor metastasis, indicates an inhibition of angiogenesis ator near the tumor site. Inhibition of non-pathogenic angiogenesis canalso be inferred from, for example, fat loss or a reduction incholesterol levels upon administration of the siRNA.

It is understood that the siRNA can degrade the target mRNA (and thusinhibit angiogenesis) in substoichiometric amounts. Without wishing tobe bound by any theory, it is believed that the siRNA causes degradationof the target mRNA in a catalytic manner. Thus, compared to standardanti-angiogenic therapies, significantly less siRNA needs to bedelivered at or near the site of neovascularization to have atherapeutic effect.

One skilled in the art can readily determine an effective amount of thesiRNA to be administered to a given subject, by taking into accountfactors such as the size and weight of the subject; the extent of theneovascularization or disease penetration; the age, health and sex ofthe subject; the route of administration; and whether the administrationis regional or systemic. Generally, an effective amount of the siRNAcomprises an intercellular concentration at or near theneovascularization site of from about 1 nanomolar (nM) to about 100 nM,preferably from about 2 nM to about 50 nM, more preferably from about2.5 nM to about 10 nM. It is contemplated that greater or lesser amountsof siRNA can be administered.

The present methods can be used to inhibit angiogenesis which isnon-pathogenic; i.e., angiogenesis which results from normal processesin the subject. Examples of non-pathogenic angiogenesis includeendometrial neovascularization, and processes involved in the productionof fatty tissues or cholesterol. Thus, the invention provides a methodfor inhibiting non-pathogenic angiogenesis, e.g., for controlling weightor promoting fat loss, for reducing cholesterol levels, or as anabortifacient.

The present methods can also inhibit angiogenesis which is associatedwith an angiogenic disease; i.e., a disease in which pathogenicity isassociated with inappropriate or uncontrolled angiogenesis. For example,most cancerous solid tumors generate an adequate blood supply forthemselves by inducing angiogenesis in and around the tumor site. Thistumor-induced angiogenesis is often required for tumor growth, and alsoallows metastatic cells to enter the bloodstream.

Other angiogenic diseases include diabetic retinopathy, age-relatedmacular degeneration (ARMD), psoriasis, rheumatoid arthritis and otherinflammatory diseases. These diseases are characterized by thedestruction of normal tissue by newly formed blood vessels in the areaof neovascularization. For example, in ARMD, the choroid is invaded anddestroyed by capillaries. The angiogenesis-driven destruction of thechoroid in ARMD eventually leads to partial or full blindness.

Preferably, an siRNA is used to inhibit the growth or metastasis ofsolid tumors associated with cancers; for example breast cancer, lungcancer, head and neck cancer, brain cancer, abdominal cancer, coloncancer, colorectal cancer, esophagus cancer, gastrointestinal cancer,glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma,ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma,Wilm's tumor, multiple myeloma; skin cancer (e.g., melanoma), lymphomasand blood cancer.

More preferably, an siRNA is used to inhibit choroidalneovascularization in age-related macular degeneration.

For treating angiogenic diseases, the siRNA can administered to asubject in combination with a pharmaceutical agent which is differentfrom the present siRNA. Alternatively, the siRNA can be administered toa subject in combination with another therapeutic method designed totreat the angiogenic disease. For example, the siRNA can be administeredin combination with therapeutic methods currently employed for treatingcancer or preventing tumor metastasis (e.g., radiation therapy,chemotherapy, and surgery). For treating tumors, the siRNA is preferablyadministered to a subject in combination with radiation therapy, or incombination with chemotherapeutic agents such as cisplatin, carboplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

In the present methods, the present siRNA can be administered to thesubject either as naked siRNA, in conjunction with a delivery reagent,or as a recombinant plasmid or viral vector which expresses the siRNA.

Suitable delivery reagents for administration in conjunction with thepresent siRNA include the Mirus Transit TKO lipophilic reagent;lipofectin; lipofectamine; cellfectin; or polycations (e.g.,polylysine), or liposomes. A preferred delivery reagent is a liposome.

Liposomes can aid in the delivery or the siRNA to a particular tissue,such as retinal or tumor tissue, and can also increase the bloodhalf-life of the siRNA. Liposomes suitable for use in the invention areformed from standard vesicle-forming lipids, which generally includeneutral or negatively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of factors such as the desired liposome size and half-lifeof the liposomes in the blood stream. A variety of methods are known forpreparing liposomes, for example as described in Szoka et al. (1980),Ann. Rev. Biophys. Bioeng. 9: 467; and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which areherein incorporated by reference.

Preferably, the liposomes encapsulating the present siRNA comprises aligand molecule that can target the liposome to a particular cell ortissue at or near the site of angiogenesis. Ligands which bind toreceptors prevalent in tumor or vascular endothelial cells, such asmonoclonal antibodies that bind to tumor antigens or endothelial cellsurface antigens, are preferred.

Particularly preferably, the liposomes encapsulating the present siRNAare modified so as to avoid clearance by the mononuclear macrophage andreticuloendothelial systems, for example by havingopsonization-inhibition moieties bound to the surface of the structure.In one embodiment, a liposome of the invention can comprise bothopsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer which significantly decreases the uptakeof the liposomes by the macrophage-monocyte system (“MMS”) andreticuloendothelial system (“RES”); e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is herein incorporated byreference. Liposomes modified with opsonization-inhibition moieties thusremain in the circulation much longer than unmodified liposomes. Forthis reason, such liposomes are sometimes called “stealth” liposomes.

Stealth liposomes are known to accumulate in tissues fed by porous or“leaky” microvasculature. Thus, target tissue characterized by suchmicrovasculature defects, for example solid tumors, will efficientlyaccumulate these liposomes; see Gabizon, et al. (1988), P.N.A.S., USA,18: 6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation inthe liver and spleen. Thus, liposomes of the invention that are modifiedwith opsonization-inhibition moieties can deliver the present siRNA totumor cells.

Opsonization inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcoholand polyxylitol to which carboxylic or amino groups are chemicallylinked, as well as gangliosides, such as ganglioside GM₁. Copolymers ofPEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are alsosuitable. In addition, the opsonization inhibiting polymer can be ablock copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. The opsonizationinhibiting polymers can also be natural polysaccharides containing aminoacids or carboxylic acids, e.g., galacturonic acid, glucuronic acid,mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginicacid, carrageenan; aminated polysaccharides or oligosaccharides (linearor branched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups.

Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes.”

The opsonization inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Recombinant plasmids which express siRNA are discussed above. Suchrecombinant plasmids can also be administered directly or in conjunctionwith a suitable delivery reagent, including the Mirus Transit LT1lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations(e.g., polylysine) or liposomes. Recombinant viral vectors which expresssiRNA are also discussed above, and methods for delivering such vectorsto an area of neovascularization in a patient are within the skill inthe art.

The siRNA can be administered to the subject by any means suitable fordelivering the siRNA to the cells of the tissue at or near the area ofneovascularization. For example, the siRNA can be administered by genegun, electroporation, or by other suitable parenteral or enteraladministration routes.

Suitable enteral administration routes include oral, rectal, orintranasal delivery.

Suitable parenteral administration routes include intravascularadministration (e.g. intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissueadministration (e.g., peri-tumoral and intra-tumoral injection,intra-retinal injection or subretinal injection); subcutaneous injectionor deposition including subcutaneous infusion (such as by osmoticpumps); direct (e.g., topical) application to the area at or near thesite of neovascularization, for example by a catheter or other placementdevice (e.g., a corneal pellet or a suppository, eye-dropper, or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Suitable placement devices include the ocular implantsdescribed in U.S. Pat. Nos. 5,902,598 and 6,375,972, and thebiodegradable ocular implants described in U.S. Pat. No. 6,331,313, theentire disclosures of which are herein incorporated by reference. Suchocular implants are available from Control Delivery Systems, Inc.(Watertown, Mass.) and Oculex Pharmaceuticals, Inc. (Sunnyvale, Calif.).

In a preferred embodiment, injections or infusions of the siRNA aregiven at or near the site of neovascularization. More preferably, thesiRNA is administered topically to the eye, e.g. in liquid or gel formto the lower eye lid or conjunctival cul-de-sac, as is within the skillin the art (see, e.g., Acheampong A A et al, 2002, Drug Metabol. andDisposition 30: 421-429, the entire disclosure of which is hereinincorporated by reference).

Typically, the siRNA is administered topically to the eye in amounts offrom about 5 microliters to about 75 microliters, for example from about7 microliters to about 50 microliters, preferably from about 10microliters to about 30 microliters. It is understood that topicalinstillation in the eye of siRNA in volumes greater than 75 microliterscan result in loss of siRNA from the eye through spillage and drainage.Thus, it is preferable to administer a high concentration of siRNA(e.g., 100-1000 nM) in as small a volume as possible.

A particularly preferred parenteral administration route is intraocularadministration. It is understood that intraocular administration of thepresent siRNA can be accomplished by injection or direct (e.g., topical)administration to the eye, as long as the administration route allowsthe siRNA to enter the eye. In addition to the topical routes ofadministration to the eye described above, suitable intraocular routesof administration include intravitreal, intraretinal, subretinal,subtenon, peri- and retro-orbital, trans-corneal and trans-scleraladministration. Such intraocular administration routes are within theskill in the art; see, e.g., and Acheampong A A et al, 2002, supra; andBennett et al. (1996), Hum. Gene Ther. 7: 1763-1769 and Ambati J et al.,2002, Progress in Retinal and Eye Res. 21: 145-151, the entiredisclosures of which are herein incorporated by reference. In anotherpreferred embodiment, the siRNA is administered by intravitrealinjection.

The siRNA can be administered in a single dose or in multiple doses.Where the administration of the siRNA is by infusion, the infusion canbe a single sustained dose or can be delivered by multiple infusions.Injection of the agent directly into the tissue is at or near the siteof neovascularization preferred. Multiple injections of the agent intothe tissue at or near the site of neovascularization are particularlypreferred.

One skilled in the art can also readily determine an appropriate dosageregimen for administering the siRNA to a given subject. For example, thesiRNA can be administered to the subject once, such as by a singleinjection or deposition at or near the neovascularization site.Alternatively, the siRNA can be administered to a subject multiple timesdaily or weekly. For example, the siRNA can be administered to a subjectonce weekly for a period of from about three to about twenty-eightweeks, more preferably from about seven to about ten weeks. In apreferred dosage regimen, the siRNA is injected at or near the site ofneovascularization (e.g., intravitreally) once a week for seven weeks.It is understood that periodic administrations of the siRNA for anindefinite length of time may be necessary for subjects suffering from achronic neovascularization disease, such as wet ARMD or diabeticretinopathy.

Where a dosage regimen comprises multiple administrations, it isunderstood that the effective amount of siRNA administered to thesubject can comprise the total amount of siRNA administered over theentire dosage regimen.

The siRNA are preferably formulated as pharmaceutical compositions priorto administering to a subject, according to techniques known in the art.Pharmaceutical compositions of the present invention are characterizedas being at least sterile and pyrogen-free. As used herein,“pharmaceutical formulations” include formulations for human andveterinary use. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art, for example as described inRemington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), the entire disclosure of which is hereinincorporated by reference.

In one embodiment, the pharmaceutical formulations comprise an siRNA(e.g., 0.1 to 90% by weight), or a physiologically acceptable saltthereof, mixed with a physiologically acceptable carrier medium.Preferred physiologically acceptable carrier media are water, bufferedwater, saline solutions (e.g., normal saline or balanced salinesolutions such as Hank's or Earle's balanced salt solutions), 0.4%saline, 0.3% glycine, hyaluronic acid and the like.

Pharmaceutical compositions can also comprise conventionalpharmaceutical excipients and/or additives. Suitable pharmaceuticalexcipients include stabilizers, antioxidants, osmolality adjustingagents, buffers, and pH adjusting agents. Suitable additives includephysiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized.

For topical administration to the eye, conventional intraocular deliveryreagents can be used. For example, pharmaceutical compositions of theinvention for topical intraocular delivery can comprise saline solutionsas described above, corneal penetration enhancers, insoluble particles,petrolatum or other gel-based ointments, polymers which undergo aviscosity increase upon instillation in the eye, or mucoadhesivepolymers. Preferably, the intraocular delivery reagent increases cornealpenetration, or prolongs preocular retention of the siRNA throughviscosity effects or by establishing physicochemical interactions withthe mucin layer covering the corneal epithelium.

Suitable insoluble particles for topical intraocular delivery includethe calcium phosphate particles described in U.S. Pat. No. 6,355,271 ofBell et al., the entire disclosure of which is herein incorporated byreference. Suitable polymers which undergo a viscosity increase uponinstillation in the eye include polyethylenepolyoxypropylene blockcopolymers such as poloxamer 407 (e.g., at a concentration of 25%),cellulose acetophthalate (e.g., at a concentration of 30%), or alow-acetyl gellan gum such as Gelrite® (available from CP Kelco,Wilmington, Del.). Suitable mucoadhesive polymers include hydrocolloidswith multiple hydrophilic functional groups such as carboxyl, hydroxyl,amide and/or sulfate groups; for example, hydroxypropylcellulose,polyacrylic acid, high-molecular weight polyethylene glycols(e.g., >200,000 number average molecular weight), dextrans, hyaluronicacid, polygalacturonic acid, and xylocan. Suitable corneal penetrationenhancers include cyclodextrins, benzalkonium chloride, polyoxyethyleneglycol lauryl ether (e.g., Brij® 35), polyoxyethylene glycol stearylether (e.g., Brij® 78), polyoxyethylene glycol oleyl ether (e.g., Brij®98), ethylene diamine tetraacetic acid (EDTA), digitonin, sodiumtaurocholate, saponins and polyoxyethylated castor oil such as CremaphorEL.

For solid compositions, conventional nontoxic solid carriers can beused; for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of one or more siRNA. A pharmaceutical compositionfor aerosol (inhalational) administration can comprise 0.01-20% byweight, preferably 1%-10% by weight, of one or more siRNA encapsulatedin a liposome as described above, and propellant. A carrier can also beincluded as desired; e.g., lecithin for intranasal delivery.

The invention will now be illustrated with the following non-limitingexamples. The animal experiments described in Examples 4-6 and 8-9 wereperformed using the University of Pennsylvania institutional guidelinesfor the care and use of animals in research. The animal experimentdescribed in Example 10 will be performed in accordance with theStandard Operating Procedures of Sierra Biomedical, 587 Dunn Circle,Sparks, Nev., 89431.

Example 1 siRNA Transfection and Hypoxia Induction In Vitro

siRNA Design—A 19 nt sequence located 329 nt from the 5′ end of humanVEGF mRNA was chosen as a target sequence: AAACCTCACCAAGGCCAGCAC (SEQ IDNO: 51). To ensure that it was not contained in the mRNA from any othergenes, this target sequence was entered into the BLAST search engineprovided by NCBI. The use of the BLAST algorithm is described inAltschul et al. (1990), J. Mol. Biol. 215: 403-410 and Altschul et al.(1997), Nucleic Acids Res. 25: 3389-3402, the disclosures of which areherein incorporated by reference in their entirety. As no other mRNA wasfound which contained the target sequence, an siRNA duplex wassynthesized to target this sequence (Dharmacon Research, Inc.,Lafayette, Colo.).

The siRNA duplex had the following sense and antisense strands.

sense: 5′-accucaccaaggccagcacTT-3′. (SEQ ID NO: 77) antisense:5′-gugcuggccuuggugagguTT-3′. (SEQ ID NO: 78)

Together, the siRNA sense and antisense strands formed a 19 ntdouble-stranded siRNA with TT 3′ overhangs (shown in bold) on eachstrand. This siRNA was termed “Candidate 5” or “Cand5.” Other siRNAwhich target human VEGF mRNA were designed and tested as described forCand5.

An siRNA targeting the following sequence in green fluorescent protein(GFP) mRNA was used as a nonspecific control: GGCTACGTCCAGCGCACC (SEQ IDNO: 79). The siRNA was purchased from Dharmacon (Lafayette, Colo.).

siRNA Transfection and Hypoxia Induction In Vitro—Human cell lines (293;Hela and ARPE19) were separately seeded into 24-well plates in 250microliters of complete DMEM medium one day prior to transfection, sothat the cells were ˜50% confluent at the time of transfection. Cellswere transfected with 2.5 nM Cand5 siRNA, and with either no siRNA or2.5 nM non-specific siRNA (targeting GFP) as controls. Transfectionswere performed in all cell lines with the “Transit TKO Transfection”reagent, as recommended by the manufacturer (Mirus).

Twenty four hours after transfection, hypoxia was induced in the cellsby the addition of desferoxamide mesylate to a final concentration of130 micromolar in each well. Twenty four hours post-transfection, thecell culture medium was removed from all wells, and a human VEGF ELISA(R&D systems, Minneapolis, Minn.) was performed on the culture medium asdescribed in the Quantikine human VEGF ELISA protocol available from themanufacturer, the entire disclosure of which is herein incorporated byreference.

As can be seen in FIG. 1, RNAi degradation induced by Cand5 siRNAsignificantly reduces the concentration of VEGF produced by the hypoxic293 and HeLa cells. There was essentially no difference in the amount ofVEGF produced by hypoxic cells treated with either no siRNA or thenon-specific siRNA control. Similar results were also seen with humanARPE19 cells treated under the same conditions. Thus, RNA interferencewith VEGF-targeted siRNA disrupts the pathogenic up-regulation of VEGFin human cultured cells in vitro.

The experiment outlined above was repeated on mouse NIH 3T3 cells usinga mouse-specific VEGF siRNA (see Example 6 below), and VEGF productionwas quantified with a mouse VEGF ELISA (R&D systems, Minneapolis, Minn.)as described in the Quantikine mouse VEGF ELISA protocol available fromthe manufacturer, the entire disclosure of which is herein incorporatedby reference. Results similar to those reported in FIG. 1 for the humancell lines were obtained.

Example 2 Effect of Increasing siRNA Concentration on VEGF Production inHuman Cultured Cells

The experiment outlined in Example 1 was repeated with human 293, HeLaand ARPE19 cells using a range of siRNA concentrations from 10 nM to 50nM. The ability of the Cand5 siRNA to down-regulate VEGF productionincreased moderately up to approximately 13 nM siRNA, but a plateaueffect was seen above this concentration. These results highlight thecatalytic nature of siRNA-mediated RNAi degradation of mRNA, as theplateau effect appears to reflect VEGF production from the few cells nottransfected with the siRNA. For the majority of cells which had beentransfected with the siRNA, the increased VEGF mRNA production inducedby the hypoxia is outstripped by the siRNA-induced degradation of thetarget mRNA at siRNA concentrations greater than about 13 nM.

Example 3 Specificity of siRNA Targeting

NIH 3T3 mouse fibroblasts were grown in 24-well plates under standardconditions, so that the cells were ˜50% confluent one day prior totransfection. The human VEGF siRNA Cand5 was transfected into a NIH 3T3mouse fibroblasts as in Example 1. Hypoxia was then induced in thetransfected cells, and murine VEGF concentrations were measured by ELISAas in Example 1 .

The sequence targeted by the human VEGF siRNA Cand5 differs from themurine VEGF mRNA by one nucleotide. As can be seen in FIG. 2, the humanVEGF siRNA has no affect on the ability of the mouse cells toup-regulate mouse VEGF after hypoxia. These results show that siRNAinduced RNAi degradation is sequence-specific to within a one nucleotideresolution.

Example 4 In Vivo Delivery of siRNA to Murine Retinal Pigment EpithelialCells

VEGF is upregulated in the retinal pigment epithelial (RPE) cells ofhuman patients with age-related macular degeneration (ARMD). To showthat functional siRNA can be delivered to RPE cells in vivo, GFP wasexpressed in mouse retinas with a recombinant adenovirus, and GFPexpression was silenced with siRNA. The experiment was conducted asfollows.

One eye from each of five adult C57/Black6 mice (Jackson Labs, BarHarbor, Me.) was injected subretinally as described in Bennett et al.(1996), supra., with a mixture containing ˜1×10⁸ particles of adenoviruscontaining eGFP driven by the CMV promoter and 20 picomoles of siRNAtargeting eGFP conjugated with transit TKO reagent (Minis).

As positive control, the contralateral eyes were injected with a mixturecontaining ˜1×10⁸ particles of adenovirus containing eGFP driven by theCMV promoter and 20 picomoles of siRNA targeting human VEGF conjugatedwith transit TKO reagent (Mims). Expression of GFP was detected byfundus opthalmoscopy 48 hours and 60 hours after injection. Animals weresacrificed at either 48 hours or 60 hours post-injection. The eyes wereenucleated and fixed in 4% paraformaldehyde, and were prepared either asflat mounts or were processed into 10 micron cryosections forfluorescent microscopy.

No GFP fluorescence was detectable by opthalmoscopy in the eyes whichreceived the siRNA targeted to GFP mRNA in 4 out of 5 mice, whereas GFPfluorescence was detectable in the contralateral eye which received thenon-specific control siRNA. A representative flat mount analyzed byfluorescence microscopy showed a lack of GFP fluorescence in the eyewhich received. GFP siRNA, as compared to an eye that received thenon-specific control siRNA. Cryosections of another retina showed thatthe recombinant adenovirus efficiently targets the RPE cells, and whenthe adenovirus is accompanied by siRNA targeted to GFP mRNA, expressionof the GFP transgene is halted.

While there is some GFP fluorescence detectable by fluorescencemicroscopy in eyes that received siRNA targeted to GFP mRNA, thefluorescence is greatly suppressed as compared to controls that receivednon-specific siRNA. These data demonstrate that functional siRNA can bedelivered in vivo to RPE cells.

Example 5 In Viva Expression and siRNA-Induced RNAi Degradation of HumanVEGF in Murine Retinas

In order to demonstrate that siRNA targeted to VEGF functioned in vivo,an exogenous human VEGF expression cassette was delivered to mouse RPEcells via an adenovirus by subretinal injection, as in Example 4. Oneeye received Cand5 siRNA, and the contralateral eye received siRNAtargeted to GFP mRNA. The animals were sacrificed 60 hourspost-injection, and the injected eyes were removed and snap frozen inliquid N₂ following enucleation. The eyes were then homogenized in lysisbuffer, and total protein was measured using a standard Bradford proteinassay (Roche, Germany). The samples were normalized for total proteinprior to assaying for human VEGF by ELISA as described in Example 1.

The expression of VEGF was somewhat variable from animal to animal. Thevariability of VEGF levels correlated well to those observed in the GFPexperiments of Example 4, and can be attributed to some error frominjection to injection, and the differential ability of adenovirus todelivery the target gene in each animal. However, there was asignificant attenuation of VEGF expression in each eye that receivedVEGF siRNA, as compared to the eyes receiving the non-specific controlsiRNA (FIG. 4). These data indicate that the Cand5 siRNA was potent andeffective in silencing human VEGF expression in murine RPE cells invivo.

Example 6 Inhibition of Choroidal Neovascularization in the Mouse CNVModel

There is evidence that choroidal neovascularization in ARMD is due tothe upregulation of VEGF in the RPE cells. This human pathologiccondition can be modeled in the mouse by using a laser to burn a spot onthe retina (“laser photo-coagulation” or “laser induction”). During thehealing process, VEGF is believed to be up-regulated in the RPE cells ofthe burned region, leading to re-vascularization of the choroid. Thismodel is called the mouse choroidal neovascularization (“CNV”) model.

For rescue of the mouse CNV model, a mouse siRNA was designed thatincorporated a one nucleotide change from the human “Cand5” siRNA fromExample 1. The mouse siRNA specifically targeted mouse VEGF mRNA at thesequence AAACCUCACCAAAGCCAGCAC (SEQ ID NO: 80). Other siRNA that targetmouse VEGF were also designed and tested. The GFP siRNA used as anonspecific control in Example 1 was also used as a non-specific controlhere.

Twenty four hours after laser induction, one eye from each of elevenadult C57/Black6 mice (Jackson Labs, Bar Harbor, Me.) was injectedsubretinally with a mixture containing ˜1×10⁸ particles of adenoviruscontaining LacZ driven by the CMV promoter and 20 picomoles of siRNAtargeting mouse VEGF conjugated with transit TKO reagent (Mirus), as inExample 4. As a control, contralateral eyes received a mixturecontaining ˜1×10⁸ particles of adenovirus containing LacZ driven by theCMV promoter and 20 picomoles of siRNA targeting GFP conjugated withtransit TKO reagent (Mirus).

Fourteen days after the laser treatment, the mice were perfused withfluorescein and the area of neovascularization was measured around theburn spots. Areas of the burn spots in the contra-lateral eye were usedas a control. The site of neovascularization around the burn spots inanimals that received siRNA targeting mouse VEGF was, on average, ¼ thearea of the control areas. These data support the use of VEGF-directedsiRNA (also called “anti-VEGF siRNA”) for therapy of ARMD.

Example 7 Generation of an Adeno-Associated Viral Vector for Expressionof siRNA

A “cis-acting” plasmid for generating a recombinant AAV vector fordelivering an siRNA was generated by PCR based subcloning, essentiallyas described in Samulski Ret al. (1987), supra. The cis-acting plasmidwas called “pAAVsiRNA.”

The rep and cap genes of psub201 were replaced with the followingsequences in this order: a 19 nt sense RNA strand coding sequence inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter, and a 19 nt antisense RNA strand codingsequence in operable connection with a polyT termination sequence underthe control of a human U6 RNA promoter. A schematic representation ofpAAVsiRNA is given if FIG. 5.

A recombinant AAV siRNA vector was obtained by transfecting pAAVsiRNAinto human 293 cells previously infected with El-deleted adenovirus, asdescribed in Fisher K J et al. (1996), supra. The AAV rep and capfunctions were provided by a trans-acting plasmid pAAV/Ad as describedin Samulski R et al. (1989), supra. Production lots of the recombinantAAV siRNA vector were titered according to the number of genomecopies/ml, as described in Fisher K J et al. (1996), supra.

Example 8 VEGF-Directed siRNA Inhibits Experimental ChoroidalNeovascularization

The ability of murine VEGF-directed siRNA to inhibit experimentallaser-induced choroidal neovascularization (CNV) in mice was tested asfollows.

The retinas of adult female C57BL/6 mice were laser photocoagulatedusing an 810 nm diode laser (75 um, 140 mw, 0.10 seconds) (OcuLight Six;IRIS Medical, Mountain View, Calif.). Three laser spots were applied toboth eyes of each mouse. Thirty-six hours following laserphotocoagulation, an siRNA targeted to mouse VEGF (“mVEGF1.siRNA”) wasdelivered subretinally or intravitreally to one eye of each mouse. Forsubretinal injection, the siRNA was conjugated with Transit TKOtransfection reagent (Mirus) and mixed with recombinant adenovirus(rAdenovirus). For intravitreal injection, the siRNA was delivered inthe absence of transfection reagent and rAdenovirus. As a control, thecontralateral eyes of each mouse received subretinal or intravitrealinjections of identical formulations with an siRNA targeted to GFP(“GFP1.siRNA”), which has no homology to mouse VEGF.

Fourteen days following laser treatment, all animals were perfused withhigh molecular weight FITC-dextran, choroidal flat mounts were preparedas described above, and the flat mounts were photographed and analyzedmicroscopically in a masked fashion. The area of CNV in each flat mountwas measured with Openlab software (Improvision, Boston, Mass.). Themean areas of CNV in eyes treated with mVEGF1.siRNA were significantlysmaller than those areas from GFP1.siRNA-treated eyes for bothsubretinal (FIG. 6A; P<0.003) and intravitreal (FIG. 6B; P<0.04)delivery.

In a second experiment, the retinas of adult female C57BL/6 mice werelaser photocoagulated as described above, and the animals were dividedinto control and test groups. One day following laser photocoagulation,phosphate buffered saline was delivered intravitreally to the animals ofthe control group, which were perfused with dextran-fluorescein 14 daysafter laser treatment. Choroidal flat mounts were then prepared and theareas of CNV in each flat mount were measured as above.

Fourteen days following laser photocoagulation, mVEGF 1.siRNA wasdelivered by intravitreal injection into one eye of each mouse in thetest group. Contralateral eyes were injected with GFP1.siRNA as acontrol. The test group animals were perfused with high molecular weightdextran-fluorescein 21 days after laser treatment. Choroidal flat mountswere then prepared and the areas of CNV in each flat mount weremeasured, as above.

In this latter experiment, the anti-VEGF siRNA was administered duringCNV growth, as opposed to before CNV growth, and thus is morerepresentative of the condition of human patients presenting with wetAMD. As can be seen from FIG. 6, the mean areas of CNV inmVEGF1.siRNA-treated eyes were significantly smaller than those areasmeasured in GFP1.siRNA-treated eyes (FIG. 6C; P<0.05). The mean areas ofCNV in mVEGF1.siRNA-treated eyes at day 21 and control (“PBS”) eyes atday 14 were not significantly different (FIG. 6C; P=0.469).

The results of these experiments indicate that age-related maculardegeneration can be treated with anti-VEGF siRNA.

Example 9 In Vivo RNA Interference of Human VEGF Induced by Anti-VEGFsiRNA in Murine RPE Cells

The ability of Cand5 siRNA to induce RNAi of VEGF in vivo over time wasevaluated as follows.

AAV.CMV.VEGF, which expresses human VEGF from an adeno-associated viralvector, was generously provided by Dr. A. Auricchio. AAV.CMV.VEGF wasinjected subretinally and bilaterally in eyes of live C57B116 mice.Twenty-eight days after injection of AAV.CMV.VEGF, Cand5 siRNA wasdelivered by intravitreal injection into one eye and control GFP1.siRNAwas delivered by intravitreal injection in the contralateral eye of eachanimal.

At day 0 (pre-siRNA injection), and at 6, 10 and 14 days after siRNAinjection, the mice were sacrificed and the eyes were snap frozen inliquid nitrogen following enucleation. The eyes were then homogenized inlysis buffer (Roche, Basel, Switzerland), and total protein was measuredusing a Bradford assay, as in Example 5 above. Two mice were used forthe 0 day time point (n=2), and three mice each were used for the 6, 10and 14 day time points (n=3). The samples were normalized for totalprotein prior to assaying for human VEGF by ELISA, according to themanufacturer's recommendations (R&D systems, Minneapolis, Minn.).Percent of VEGF (% VEGF) for each mouse was calculated as theconcentration of VEGF (“[VEGF]”) in the eye injected with Cand5 dividedby the [VEGF] in the eye injected with GFP1.siRNA, multiplied by 100.

As can be seen from FIG. 7, a single injection of Cand5 induced anRNAi-mediated decrease in VEGF levels of approximately 70% by day 6post-siRNA injection, with a reduction in VEGF production ofapproximately 35% continuing through at least day 14 post-siRNAinjection. These results indicate that siRNA directed against human VEGFis capable of inducing RNAi of human VEGF in vivo for a sustained periodof time.

Example 10 In Vivo RNA Interference of VEGF in Monkeys with Anti-VEGFsiRNA

The objectives of this study were to determine the safety and efficacyof Cand5 when administered by single intravitreal injection to malecynomolgus monkeys following induction of CNV. Cand5 was administered inthe vehicle control article to naive male cynomolgus monkeys in thefollowing dose levels: 0 mg/eye (control), 0.07 mg/eye, 0.18 mg/eye,0.35 mg/eye and, and 0.70 mg/eye.

CNV was induced by laser treatment to the maculae of both eyes of eachanimal, and the doses of Cand5 were given shortly following lasertreatment. The animals were evaluated for changes in clinical signs,body weight and ocular condition (extensive ophthalmic examinations,electroretinography and tonometry). Fluorescein angiography wasperformed and blood samples were collected. At the end of the study (Day44), all animals were euthanized and a complete gross necropsy wasperformed. Selected tissues were collected and preserved forhistopathologic evaluation.

No adverse systemic or local (ocular) effects of Cand5 were detectedwhen monkeys were administered a single intravitreal injection into botheyes at doses up to 0.70 mg/eye following laser lesioning of the maculaand during subsequent development of CNV.

Example 11 In Vitro RNA Interference of VEGF with Anti-VEGF siRNA inHuman Embryonic Kidney 293 Cells

Human embryonic kidney 293 cells (obtained from ATCC, Manassas, Va.)were cultured in Dulbecco's Modified Eagle Medium (DMEM; obtained fromCellgro, Herndon, Va.) with 10% fetal bovine serum (FBS; from JRHBiosciences, Lenexa, Kans.) and an antibiotic-antimycotic reagent, usedfor the prevention of cell culture growth contaminants (from Gibco,Carlsbad, Calif.).

siRNAs were synthesized by Integrated DNA Technologies (Coralville,Iowa). The siRNA target sequences are shown in Table 2. An additionalsiRNA was used in this study that targets the gene of enhanced greenfluorescent protein (EGFP) as a negative control.

TABLE 2 GC Nucleotide Name Content Start Site Target Sequence 5′-3′hVEGF#1 58%  92 aaggaggagggcagaatcatc (SEQ ID NO: 81) hVEGF#2 42% 124aagttcatggatgtctatcag (SEQ ID NO: 47) hVEGF#3 58% 162aatcgagaccctggtggacat (SEQ ID NO: 48) hVEGF#4 42% 301aacatcaccatgcagattatg (SEQ ID NO: 50) hVEGF#5 58% 338aaggccagcacataggagaga (SEQ ID NO: 52) hVEGF#6 42% 380aatgtgaatgcagaccaaaga (SEQ ID NO: 82) hVEGF#7 37% 396aaagaaagatagagcaagaca (SEQ ID NO: 56) hVEGF#8 32% 450aaagcatttgtttgtacaaga (SEQ ID NO: 83) hVEGF#9 42% 467aagatccgcagacgtgtaaat (SEQ ID NO: 84) hVEGF#10 53% 498aaacacacactcgcgttgcaa (SEQ ID NO: 85) Cand5 63% 328aaacctcaccaaggccagcac (SEQ ID NO: 51)

siRNA Transfection and Hypoxia Induction In Vitro. Human 293 cells werecultured in 24 well plates at 37° C. with 5% CO2 overnight. The nextday, transfections were performed when cells were about 50%-70%confluent. Cells were transfected with siRNAs directed against humanVEGF. siRNAs were mixed in a CaPi reagent and added to 20 μl of 250 mMCaCl₂ solution. The siRNA/CaCl₂ mixture was added drop-wise to 20 μl of2× Hanks Balanced Salt Solution (HBS), while mixing by vortex. ThesiRNA/CaCl₂/HBS complex was added directly to the medium in each well(300 μL/well). After a 4-hour incubation at 37° C., the medium wasremoved, and the cells were further incubated with 10% DMSO-containingserum-free medium (300 μL/well at room temperature for 1-2 minutes).This medium was then removed, and the cells were fed again with growthmedium (500 μL/well). Negative controls included transfection reagentlacking siRNA and nonspecific siRNA (EGFP1 siRNA). For screeningexperiments siRNAs were used at a concentration of 25 nM. For doseresponse experiments, siRNAs were used at concentrations of 1 nM, 5 nMand 25 nM. Hypoxia was induced with desferrioxamine at a finalconcentration of 130 uM 4 hours after transfection was performed.Desferrioxamine mimics a hypoxic state, as it is proposed to disruptnormal oxygen-sensing pathways in mammalian cells by inhibitingheme-Fe2+ interactions.

VEGF Protein Quantification. Approximately 48 hours post transfection,the supernatant was removed from all wells and a human VE GF ELISA (R &D systems, Minneapolis, Minn.) was performed on the 293 cells asdescribed in the Quantikine human VEGF ELISA protocol. VEGF-specificAntibody was added to each well causing color development in proportionto the amount of VEGF bound to the plate. ELISA results were read on anAD340 plate reader at 450 nm (Beckman Coulter).

Results. Human VEGF siRNAs Suppresses Hypoxia-Induced Up-regulation ofHuman VEGF Protein in 293 Cells. Human VEGF was upregulated by thedesferrioxamine-mediated induction of hypoxia. Readings of OD 450 nmreflected the human VEGF protein levels in cell samples. Thehypoxia-induced increase of hVEGF protein levels were significantlyreduced in cells transfected with all of the human VEGF siRNAs (FIG. 8).No effect on hVEGF levels were observed with transfections withnonspecific siRNA (EGFP siRNA) or mock transfections without siRNA. Doseresponse studies were performed on Candy, hVEGF#1, hVEGF#2, hVEGF#3,hVEGF#4, hVEGF#6 and hVEGF#7 (FIG. 9).

1. An isolated siRNA comprising of a duplex of a first RNA strand and asecond RNA strand, said first RNA strand comprising a nucleotidesequence identical to a target sequence of about 19 to about 25contiguous nucleotides in said human VEGF mRNA.
 2. The siRNA of claim 1,wherein said human VEGF mRNA is selected from VEGF₁₂₁ mRNA (SEQ ID NO:2); VEGF₁₆₅ mRNA (SEQ ID NO: 3); VEGF₁₈₉ mRNA (SEQ ID NO: 4) and VEGF₂₀₆mRNA (SEQ ID NO: 5).
 3. The siRNA of claim 1, wherein the first andsecond RNA strands forming the RNA duplex are covalently linked by asingle-stranded hairpin.
 4. The siRNA of claim 1, wherein the siRNAfurther comprises non-nucleotide material.
 5. The siRNA of claim 1,wherein the first and second RNA strands are stabilized against nucleasedegradation.
 6. The siRNA of claim 1, further comprising a 3′ overhang.7. The siRNA of claim 6, wherein the 3′ overhang comprises from 1 toabout 6 nucleotides.
 8. The siRNA of claim 6, wherein the 3′ overhangcomprises about 2 nucleotides.
 9. The siRNA of claim 1, wherein thesense RNA strand comprises a first 3′ overhang, and the antisense RNAstrand comprises a second 3′ overhang.
 10. The siRNA of claim 9, whereinthe first and second 3′ overhangs each comprise from 1 to about 6nucleotides.
 11. The siRNA of claim 9, wherein the first 3′ overhangcomprises a dinucleotide and the second 3′ overhang comprises adinucleotide.
 12. The siRNA of claim 11, where the dinucleotidecomprising the first and second 3′ overhangs is dithymidylic acid (TT)or diuridylic acid (uu).
 13. The siRNA of claim 6, wherein the 3′overhang is stabilized against nuclease degradation.
 14. Apharmaceutical composition comprising a siRNA and a pharmaceuticallyacceptable carrier, said siRNA comprised of a duplex of a first RNAstrand and a second RNA strand, said first RNA strand comprising anucleotide sequence identical to a target sequence of about 19 to about25 contiguous nucleotides in said human VEGF mRNA.
 15. Thepharmaceutical composition of claim 14, wherein said human VEGF mRNA isselected from VEGF₁₂₁ mRNA (SEQ ID NO: 2); VEGF₁₆₅ mRNA (SEQ ID NO: 3);VEGF₁₈₉ mRNA (SEQ ID NO: 4) and VEGF₂₀₆ mRNA (SEQ ID NO: 5).
 16. Thepharmaceutical composition of claim 14, wherein the first and second RNAstrands are stabilized against nuclease degradation.
 17. Thepharmaceutical composition of claim 14, further comprising at least one3′ overhang.
 18. The pharmaceutical composition of claim 17, wherein theat least one 3′ overhang comprises about 2 nucleotides.
 19. Thepharmaceutical composition of claim 17, where the at least one 3′overhang comprises a dithymidylic acid (TT) or diuridylic acid (uu). 20.The pharmaceutical composition of claim 14, wherein the sense RNA strandcomprises a first 3′ overhang, and the antisense RNA strand comprises asecond 3′ overhang.